jfbc_337 856..

868

DOI: 10.1111/j.1745-4514.2010.00337.x

ANTIHYPERGLYCEMIC EFFECT OF AQUEOUS EXTRACT OF

SEA BUCKTHORN (HIPPOPHAE RHAMNOIDES L.) SEED
RESIDUES IN STREPTOZOTOCIN-TREATED AND HIGH
FAT-DIET-FED RATS

WEN ZHANG, JINGJING ZHAO, XINGLEI ZHU, XIUYUAN ZHUANG,

XIUFENG PANG, JIESI WANG and WEIJING QU1

School of Life Science

East China Normal University
3663 Zhongshang North Road
Shanghai 200062, China

Accepted for Publication November 10, 2008

ABSTRACT

The present study was designed to investigate the antihyperglycemic

activity of the aqueous extract of sea buckthorn (Hippophae rhamnoides L.)
seed residues (ASSR) in streptozotocin (STZ) and high-fat diet (HFD)-induced
type 2 diabetic rats.
Male Sprague-Dawley rats were divided into a normal control group
(control), diabetic group and diabetic group supplemented with ASSR. Type 2
diabetes was induced by intravenous injection of low-dose STZ (25 mg/kg
b.w.) and feeding with HFD for 10 weeks. Vehicle (distilled water) and ASSR
(400 mg/kg b.w.) were orally administered once a day for 6 weeks. Our results
showed that administration of ASSR significantly lowered the body weight,
serum glucose, total cholesterol and low-density lipoprotein cholesterol levels
in diabetic rats. Meanwhile, ASSR also significantly increased insulin sensi-
tivity index in diabetic rats. These results suggest that ASSR might exhibit
hypoglycemic and hypolipidemic effects in the STZHFD-induced type 2 dia-
betic rats.

PRACTICAL APPLICATIONS

Sea buckthorn (Hippophae rhamnoides L.) bush, bearing delicious and

nutritious orange-colored berries, is locally called Shaji in China. The berry
of sea buckthorn is one of the edible crude herbs that have been in use in

1
Corresponding author. TEL: +86-21-62232019; FAX: +86-21-62233754; EMAIL: wjqu@
bio.ecnu.edu.cn

Journal of Food Biochemistry 34 (2010) 856868.

856 2010, Wiley Periodicals, Inc.
 HYPOGLYCEMIC EFFECT OF SEA BUCKTHORN SEED RESIDUES 857

Tibetan and Mongolian traditional medicines for a long time. In addition to

medicinal use, the berries are processed into various products such as juice and
marmalade, and are used for flavoring of dairy products. As a part of sea
buckthorn berry, its seed is a by-product of the berry processing industry and
also contains many bioactive substances.
It was demonstrated in this study that ASSR was of hypoglycemic and
hypolipidemic properties in experimental type 2-like diabetic rats. Therefore,
seed residues of sea buckthorn and its aqueous extract could be added as
supplement in health-care food or drugs or combined with other hypoglycemic
drugs.

INTRODUCTION

Hippophae rhamnoides L. is commonly known as sea buckthorn (family:

Elaeagnaceae) growing wildly in Asia and Europe. The plant is a winter-hardy,
drought-resistant and thorny, nitrogen-fixing deciduous shrub (Sabir et al.
2003; Ercisli et al. 2007). Sea buckthorn is rich in many phytochemicals,
such as fatty oil, lipids, flavones, carotenoids, tocopherols, sterols, tannins and
ascorbic acid, etc. (Yang et al. 1998; Guliyev et al. 2004). As a Tibetan and
Mongolian traditional medicine, sea buckthorn has been reported to possess
immunomodulatory, antioxidant, antibacterial, anti-inflammatory and cardio-
protective properties (Cheng and Li 1992; Gao et al. 2000; Suleyman et al.
2001; Ganjua et al. 2005). The berries of sea buckthorn can be used in the
treatment of several diseases, such as cardiovascular diseases, cancer and acute
mountain sickness, while pulp and seed oils, juice, leaves and bark have been
used to treat high blood lipid symptoms, gingivitis, eye and skin ailments,
cardiovascular diseases, thrombosis and platelet aggregation (Yang et al.
2000; Negi et al. 2005; Saggu et al. 2007).
As the active principle of sea buckthorn seed, the oil content of seeds
ranges from 12 to 13% and has been used in folk medicine (Zadernowski et al.
2002). But after oil is removed, the remaining residues of seed, a by-product
of the seed processing industry, have not been exploited as yet. Earlier studies
in our laboratory showed that the seed residues (oil removed) extract of sea
buckthorn was of hypoglycemic and hypolipidemic properties in streptozoto-
cin (STZ)-induced type 1 diabetic rats when administered orally (Cao et al.
2005). Accordingly, the current study was performed to further evaluate the
antidiabetic action of aqueous extract of sea buckthorn seed residues (ASSR)
in a different type 2-like diabetic animal model, the low-dose STZ and high-fat
diet (HFD)-induced type 2 diabetic rats.
858 W. ZHANG ET AL.

MATERIALS AND METHODS

Materials
STZ was purchased from Sigma Chemical Co. (St. Louis, MO). Blood
glucose, triglyceride (TG), total cholesterol (TC), free fatty acids (FFA),
high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein
cholesterol (LDL-C) diagnosis kits were obtained from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China).

Preparation of Plant Extracts

The seed residues of H. rhamnoides L. were obtained from Inner Mon-
golia Yuhangren Hi-Tech Industrial Co., Ltd. (Hohhot, Inner Mongolia,
China). The corresponding seeds were collected in Chifeng, Inner Mongolia
and the voucher specimen (No. Wang S.Y. 2006001) was deposited in the
Herbarium of East China Normal University (Shanghai, China) herbarium.
One hundred grams of powdered seed residues were boiled twice (1 h each) in
water (1:10 w/v). After filtration, the extract was concentrated with a vacuum
rotary evaporator and was freeze-dried.

Animals and Diet

Male Sprague-Dawley rats 200220 g were purchased from Super-B &
K, Ltd. (Shanghai, China) and housed in an air-conditioned room with con-
trolled temperature (2325C) and automatic lighting (on a 12-h light/12-h dark
cycle). They were fed on a standard pellet diet (obtained from Shanghai SLAC
Laboratory Animal Co. Ltd., Shanghai, China) and water ad libitum.
The normal chow diets contained 9.2% water, 22.1% crude protein,
5.28% crude fat, 5.20% crude ash, 4.12% crude fiber, 1.24% calcium, 0.92%
phosphorous, 0.72% mixture of dl-methionine and leucine, 1.34% lysine and
52.0% nitrogen free extract. HFDs contained 19.75% lard, 7.79 % sucrose and
72.46% normal chow diets.
The license number of using experimental animals is SYXK(SH) 2004-
0001, and all protocols of animal maintenance and handling were in accor-
dance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals (National Research Council 1985).

Experimental Induction of Type 2-Like Diabetes in Rats

Type 2-like diabetes in rats was induced by a modification of Xies
procedure (Xie et al. 2006) and this diabetic model is associated with insulin
resistance. Briefly, male SD rats were injected intravenously with a low-dose
STZ (25 mg/kg b.w.) freshly dissolved in citrate buffer (0. 1 mol/L, pH 4.5);
 HYPOGLYCEMIC EFFECT OF SEA BUCKTHORN SEED RESIDUES 859

simultaneously, HFD were fed to these rats. Ten weeks later, blood samples
were obtained from the tail vein for assay of fasting serum glucose. According
to the previous study (Sahin et al. 2007), the STZHFD-treated rats that
exhibited fasting glucose levels greater than 140 mg/dL (7.78 mmol/L) were
considered as neonatal STZ diabetic resembling type 2 diabetes mellitus in
humans. So the rats with fasting serum glucose concentrations between 8 and
12 mmol/L were chosen for experiment.
Experimental Procedure
Rats were divided into three groups of 10 rats in each: Group 1, normal
control rats (distilled water treated); Group 2, STZHFD-induced diabetic rats
(distilled water treated); Group 3, STZHFD-induced diabetic rats supple-
mented with ASSR at a dose of 400 mg/kg b.w. ASSR and distilled water were
administered intragastrically once a day lasting for 6 weeks.
During experimental period, serum glucose concentrations and body
weight were monitored every 2 weeks. At the end of the experiments, all
rats were anesthetized quickly using chloral hydrate (450 mg/kg) after a 12-h
fast. Blood samples taken from the abdominal aorta were centrifuged at
3,000 rpm for 15 min at 4C and serum was collected for biochemical analysis.
All samples were stored at -70C until analysis.
Biochemical Analysis
Serum glucose, TGs, TC, FFA, HDL-C and LDL-C levels were deter-
mined by using commercial kits purchased from Nanjing Jiancheng Bioengi-
neering Institute.
Serum insulin contents were determined by a double-antibody radioim-
munoassay method, using rat insulin standards (Novo Biolabs, Bagsbaevard,
Denmark) and Linco Research (St. Louis, MO) antibodies (guinea pig anti-rat
insulin antibody, goat anti-guinea pig immunoglobulin [lg] G serum antibody
and guinea pig carrier lgG) according to manufactures instructions (Uchida
et al. 1997). The units of results were expressed as pmol/L in serum. Insulin
sensitivity index (ISI) was calculated according to the formula described by Li
and Pan (1998):

ISI = Ln [1 ( FPG FINS)]

FPG (mM) represents fasting plasma glucose and FINS (pmol/L) repre-
sents fasting plasma insulin.
Statistical Analyses
All data are presented as the mean standard error. The data were
evaluated by a one-way analysis of variance using the SPSS program (SPSS,
860 W. ZHANG ET AL.

Chicago, IL), and the differences between the means were assessed using
Duncans multiple range test. P < 0.05 was considered statistically different.

RESULTS

Effect of ASSR on Serum Glucose in STZHFD-Induced Diabetic Rats

The changes in the serum glucose concentrations of different experimen-
tal groups are illustrated in Fig. 1. The STZHFD-induced diabetic rats exhib-
ited a significant (P < 0.01) increase in serum glucose level compared with the
normal controls. ASSR caused a significant (P < 0.01) hypoglycemic effect
after daily oral administration at a dose of 400 mg/kg b.w. for 2 weeks com-
pared with the vehicle-treated diabetic group. Similarly, the long-term admin-
istration of ASSR for 4 and 6 weeks in diabetic rats also showed a significant
(P < 0.05) serum glucose-lowering action.
Effect of ASSR on Body Weight in STZHFD-Induced Diabetic Rats
Effect of ASSR on body weight in STZHFD-induced diabetic rats is
shown in Fig. 2. There was a significant (P < 0.05) increase in the body weight

Control

12 Diabetic
Diabetic+ASSR
Serum glucose (mmol/L)

10
*

*
8 **

** **
6 **

4
0 2 4 6
Time (weeks)

FIG. 1. EFFECT OF AQUEOUS EXTRACT OF SEA BUCKTHORN SEED RESIDUES (ASSR)

ON FASTING SERUM GLUCOSE LEVEL IN STREPTOZOTOCIN (STZ) AND HIGHFAT
DIET (HFD) DIABETIC RATS
Control, normal control rats, administered distilled water; Diabetic, STZHFD-induced diabetic rats,
administered distilled water; Diabetic + ASSR, STZHFD-induced diabetic rats supplemented with
ASSR at a dose of 400 mg/kg b.w. The fasting serum glucose levels were measured every 2 weeks.
Graph represent the mean standard error (n = 10). * P < 0.05 versus the vehicle-treated diabetic
rats (diabetic). ** P < 0.01 versus the vehicle-treated diabetic rats (diabetic).
 HYPOGLYCEMIC EFFECT OF SEA BUCKTHORN SEED RESIDUES 861

Control
Diabetic
540 Diabetic+ASSR
* *
450 *
Body weight (g)

360
270
180

90
0
0 w eek 6 w eeks

FIG. 2. THE BODY WEIGHT IN CONTROL AND STREPTOZOTOCIN (STZ) AND HIGHFAT
DIET (HFD) DIABETIC RATS
Control, normal control rats, administered distilled water; Diabetic, STZHFD-induced diabetic rats,
administered distilled water; Diabetic + aqueous extract of sea buckthorn seed residues (ASSR),
STZHFD-induced diabetic rats supplemented with ASSR at a dose of 400 mg/kg b.w. Columns
represent the mean standard error (n = 10). * P < 0.05 versus the vehicle-treated diabetic
rats (diabetic).

of STZHFD-diabetic rats compared with the normal controls. At the end of

the experiments, a significant reduction (P < 0.05) was observed in body
weight in ASSR-treated diabetic group compared with the vehicle-treated
diabetic group.
Effect of ASSR on Serum Insulin and ISI in STZHFD-Induced
Diabetic Rats
In the STZHFD-induced diabetic rats, serum insulin levels were signifi-
cantly increased (Fig. 3, P < 0.01), whereas ISI were significantly decreased
(Fig. 4, P < 0.01) compared with the normal control rats. The supplementation
of ASSR significantly increased (Fig. 4, P < 0.01) the ISI in the diabetic rats.
At the end of the experiments, there was no significant difference in serum
insulin level of experimental rats (Fig. 3).
Effect of ASSR on Serum Lipid Profiles in STZHFD-Induced
Diabetic Rats
The changes in serum lipid profiles of different experimental groups are
illustrated in Table 1; the STZHFD-induced diabetic rats exhibited significant
increase in serum TG, TC, LDL-C (P < 0.01) and FFA (P < 0.05) concentra-
tions, whereas HDL-C concentrations were markedly decreased (P < 0.01)
compared with the normal control rats. The administration of ASSR to diabetic
rats for 6 weeks resulted in the marked decrease in TC and LDL-C levels
(P < 0.01). The serum TG, FFA and HDL-C levels did not differ between the
diabetic and diabetic ASSR groups.
862 W. ZHANG ET AL.

Control
240
Diabetic
Serum insulin (pmol/L)

Diabetic+ASSR
180

**
120

60

0
0 week 6 weeks

FIG. 3. THE SERUM INSULIN LEVELS IN CONTROL AND STREPTOZOTOCIN (STZ) AND
HIGHFAT DIET (HFD) DIABETIC RATS
Control, normal control rats, administered distilled water; Diabetic, STZHFD-induced diabetic rats,
administered distilled water; Diabetic + aqueous extract of sea buckthorn seed residues (ASSR),
STZHFD-induced diabetic rats supplemented with ASSR at a dose of 400 mg/kg b.w. The serum
insulin levels were measured on week 0 and week 6. Columns represent the mean standard error
(n = 10). ** P < 0.01 compared with the vehicle-treated diabetic rats (diabetic).

Diabetic+ASSR
Diabetic **
Control 6 weeks
**

0 week
**

-7.5 -7 -6.5 -6

Insulin sensitivity index

FIG. 4. THE INSULIN SENSITIVITY INDEX IN CONTROL AND STREPTOZOTOCIN (STZ)

AND HIGHFAT DIET (HFD) DIABETIC RATS
Control, normal control rats, administered distilled water; Diabetic, STZHFD-induced diabetic rats,
administered distilled water; Diabetic + aqueous extract of sea buckthorn seed residues (ASSR),
STZHFD-induced diabetic rats supplemented with ASSR at a dose of 400 mg/kg b.w. Columns
represent the mean standard error (n = 10). ** P < 0.01 compared with the vehicle-treated
diabetic rats (diabetic).

DISCUSSION

Previous studies in our laboratory showed that seed residues (oil

removed) extract of sea buckthorn exhibited orally active hypoglycemic and
 HYPOGLYCEMIC EFFECT OF SEA BUCKTHORN SEED RESIDUES 863

TABLE 1.
EFFECT OF ASSR ON SERUM LIPID PARAMETERS IN STZHFD-INDUCED
DIABETIC RATS

TG TC HDL-C LDL-C FFA

(mmol/L) (mmol/L) (mmol/L) (mmol/L) (mmol/L)

Control 0.71 0.05** 1.46 0.15** 1.22 0.11** 0.30 0.03** 0.24 0.03*
Diabetic 1.32 0.18 2.21 0.10 0.77 0.04 1.11 0.10 0.46 0.08
Diabetic+ASSR 1.24 0.10 1.70 0.12** 0.81 0.07 0.64 0.06** 0.36 0.06

Each value represents mean standard error for 10 rats each group.
* P < 0.05, ** P < 0.01 as compared with the vehicle-treated diabetic rats (Diabetic group). Control,
normal control rats, administered distilled water; Diabetic, STZHFD-induced diabetic rats, admin-
istered distilled water; Diabetic + ASSR, STZHFD-induced diabetic rats supplemented with ASSR
at a dose of 400 mg/kg B.W.
ASSR, aqueous extract of sea buckthorn seed residues; HFD, high-fat diet; STZ, streptozotocin; TG,
triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low density-
lipoprotein cholesterol; FFA, free fatty acids.

hypolipidemic effects in normal mice (Cao et al. 2003). Further studies indi-
cated that seed residues extract has hypoglycemic, hypotriglyceridemic and
antioxidant effects in STZ-induced type 1-like diabetic rats (Cao et al. 2005).
It significantly lowered serum malondialdehyde level and increased serum
glutathione level and glutathione reductase activity.
In the present study, we used ASSR, the aqueous extract from sea buck-
thorn seed residues, to treat experimental type 2-like diabetic rats. We focused
on measuring the change of serum glucose, insulin, lipid profiles and body
weight to investigate the effect of ASSR on glucose and lipid metabolism and to
evaluate antidiabetic effect of ASSR on a rat model of type 2-like diabetes, the
low-dose STZHFD-induced type 2 diabetic rats. STZ has been widely used to
induce diabetes in rats. Although high-dose STZ severely impairs insulin
secretion mimicking type 1 diabetes mellitus, low-dose STZ has been known to
induce a mild impairment of insulin secretion characteristic of the later stage of
type 2 diabetes mellitus (Sugano et al. 2006). A diet high in fat has generally
been considered to be a predisposing factor for diabetes, hypertension and
atherosclerosis, and the effect of a HFD on glucose metabolism has been
reported in many diabetic animal models including STZ-treated rats, which
have invariably shown that HFDs cause insulin resistance (Kim et al. 2000;
Sugano et al. 2006). The type 2-like diabetic animal model induced by low-dose
STZ injection (25 mg/kg) plus HFD feeding, which simulates the metabolic
characteristics of patients with type 2 diabetes, has been recognized as type 2
diabetes model. In our experiments, the STZHFD-induced diabetic rats exhib-
ited significant increase in serum glucose, insulin, TG, TC, LDL-C (P < 0.01)
and FFA (P < 0.05) levels, whereas HDL-C levels and ISI significantly
864 W. ZHANG ET AL.

decreased (P < 0.01) compared with the normal control rats. These findings
suggested that these rats had metabolic characteristics of type 2 diabetes,
including obesity, mild hyperglycemia, dyslipidemia and insulin resistance.
With this model, antidiabetic effects of ASSR were investigated. We
measured several biomarkers in STZHFD-induced type 2-like diabetic rats
such as serum glucose, insulin and lipid profiles. The results indicated that
ASSR exhibited significant hypoglycemic and lipid-lowering effects in type
2-like diabetic rats. It markedly reduced serum glucose, TC and LDL-C levels
in diabetic rats.
Type 2 diabetes mellitus is a multifactorial disease characterized by
insulin resistance with a relative impairment in insulin secretion. The natural
history of type 2 diabetes mellitus begins with a period of insulin resistance
with augmented pancreatic insulin secretion. As the disease progresses, pan-
creatic function falters and is no longer able to meet peripheral demands. As a
result, insulin levels fail to keep up with the body requirements (Srinivasan
et al. 2005). In this study, after STZ injection and a HFD feeding, the serum
insulin levels of rats were significantly increased (P < 0.01). After 6 weeks
of ASSR administration, no significant alterations were observed in serum
insulin of experimental rats. This result suggested that the hypoglycemic
activity of ASSR observed in our study was not related to stimulation of insulin
secretion. Hence, the hypoglycemic effect might be probably brought about by
an extra pancreatic mechanism. It is known that all forms of diabetes mellitus
are due to a decrease in the circulating concentration of insulin (insulin
deficiency) or a decrease in the response of peripheral tissue to insulin (insulin
resistance). Insulin resistance profoundly contributes to the pathophysiology
of type 2 diabetes and induces reduced glucose utilization and increased
glucose production in the liver, leading to hyperglycemia (Jung et al. 2006).
Hence, besides stimulation of insulin secretion, enhancement of insulin sen-
sitivity has also been indicated as one of the mechanisms for antidiabetic
action. At present, oral therapy for type 2 diabetes relies upon insulin secre-
tagogues such as glibenclamide, and insulin sensitizers such as thiazolidinedi-
one (Kobayashi et al. 1992; Jung et al. 2007). The present study showed that
oral administration of ASSR for 6 weeks effectively increased ISI, denoting
increased insulin sensitivity in type 2-like diabetic rats. These results implied
that ASSR exerted hypoglycemic effect most likely through enhancing insulin
sensitivity rather than stimulating insulin secretion. However, the precise
mechanisms of ASSR need further investigation.
Insulin resistance, in both human and animal models, is commonly asso-
ciated with several abnormalities in the lipid metabolism, including increased
plasma FFA levels, hypertriglyceridemia and hypercholesterolemia. Improve-
ment of dyslipidemia is helpful in the enhancement of insulin sensitivity. As
shown in our research, the ASSR possessed definite hypocholesterolemic and
 HYPOGLYCEMIC EFFECT OF SEA BUCKTHORN SEED RESIDUES 865

LDL-C-lowering properties in STZHFD-induced diabetic rats after 6 weeks

of treatment. With this result, we propose that the insulin-sensitizing action of
ASSR might be due to improvement of dyslipidemia by reducing the serum
TC and LDL-C levels.
Although the pathogenesis of type 2 diabetes is not completely under-
stood, evidence is accumulating which supports the notion that type 2 diabetes
results from abnormalities in glucose and lipid metabolism. The most common
lipid abnormalities in diabetes are hypertriglyceridemia and hypercholester-
olemia. Hypertriglyceridemia is associated with metabolic consequences of
hypercoagulability, hyperinsulinemia, insulin resistance and insulin intoler-
ance (Shirwaikar et al. 2005). High level of TC is one of the major factors for
coronary heart diseases and it is well known that hyperlipidemia and the
incidences of atherosclerosis increase in diabetes (Tan et al. 2005). Lowering
of serum lipid levels through dietary or drug therapy seems to be associated
with a decrease in the risk of vascular disease and related complications
(Kumar and Murugesan 2008). The hypocholesterolemic and LDL-C-
lowering properties of ASSR observed in this investigation implied that it
would be helpful in the prevention of diabetic complications through improv-
ing dyslipidemia. In this work, the characteristic increase in body weight is
associated with STZHFD-induced diabetes. The ASSR-supplemented rats
showed an obvious weight loss in this study, which might be due to the
lipid-lowering action of this extract.
In conclusion, the ASSR exhibited hypoglycemic activity and this activity
seemed to associate with enhancing insulin sensitivity by decreasing the
elevated serum TC and LDL-cholesterol levels in experimental type 2-like
diabetic rats. Further investigations in purifying the active compounds from
the aqueous extract will be necessary to elucidate the precise mechanism of its
hypoglycemic and hypolipidemic actions.

ACKNOWLEDGMENT

The research was supported by a science foundation from the Science and
Technology Commission of Shanghai Municipality (014358009).

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